Sean D. Stocker

Water deprivation activates a glutamatergic projection from the hypothalamic paraventricular nucleus to the rostral ventrolateral medulla.

Elevated sympathetic outflow contributes to the maintenance of blood pressure in water‐deprived rats. The neural circuitry underlying this response may involve activation of a pathway from the hypothalamic paraventricular nucleus (PVH) to the rostral ventrolateral medulla (RVLM). We sought to determine whether the PVH‐RVLM projection activated by water deprivation is glutamatergic and/or contains vasopressin‐ or oxytocin‐neurophysins. Vesicular glutamate transporter 2 (VGLUT2) mRNA was detected by in situ hybridization in the majority of PVH neurons retrogradely labeled from the ipsilateral RVLM with cholera toxin subunit B (CTB; 85% on average, with regional differences). Very few RVLM‐projecting PVH neurons were immunoreactive for oxytocin‐ or vasopressin‐associated neurophysin. Injection of biotinylated dextran amine (BDA) into the PVH produced clusters of BDA‐positive nerve terminals within the ipsilateral RVLM that were immunoreactive (ir) for the VGLUT2 protein. Some of these terminals made close appositions with tyrosine‐hydroxylase‐ir dendrites (presumptive C1 cells). In water‐deprived rats (n = 4), numerous VGLUT2 mRNA‐positive PVH neurons retrogradely labeled from the ipsilateral RVLM with CTB were c‐Fos‐ir (16–40%, depending on PVH region). In marked contrast, few glutamatergic, RVLM‐projecting PVH neurons were c‐Fos‐ir in control rats (n = 3; 0–3%, depending on PVH region). Most (94% ± 4%) RVLM‐projecting PVH neurons activated by water deprivation contained VGLUT2 mRNA. In summary, most PVH neurons that innervate the RVLM are glutamatergic, and this population includes the neurons that are activated by water deprivation. One mechanism by which water deprivation may increase the sympathetic outflow is activation of a glutamatergic pathway from the PVH to the RVLM. J. Comp. Neurol. 494:673–685, 2006.

Sympathetic Response to Insulin Is Mediated by Melanocortin 3/4 Receptors in the Hypothalamic Paraventricular Nucleus

Hyperinsulinemia increases sympathetic nerve activity and contributes to cardiovascular dysfunction in obesity and diabetes. Neurons of the hypothalamic paraventricular nucleus (PVN) regulate sympathetic nerve activity through mono- and poly-synaptic connections to preganglionic neurons in the spinal cord. The purpose of the present study was to determine whether PVN neurons mediate the sympathetic response to insulin. Hyperinsulinemic-euglycemic clamps were performed in &agr;-chloralose-anesthetized, male Sprague-Dawley rats (280–420 g) by an infusion of insulin (3.75 mU/kg per min) and 50% dextrose (0.75–2.0 mL/h) for 120 minutes. At 90 minutes, insulin significantly increased lumbar sympathetic nerve activity without any change in renal sympathetic nerve activity, heart rate, or blood glucose levels. Inhibition of the PVN with bilateral injection of the GABAA receptor agonist muscimol completely reversed the sympathoexcitatory response. However, direct injection of insulin into the PVN did not alter lumbar sympathetic nerve activity, and thereby suggests that insulin activates neurons upstream of the PVN. Interestingly, the sympathetic response to insulin was eliminated by PVN injection of the melanocortin 3/4 receptor antagonist SHU9119, but was unaffected by the angiotensin II type 1 receptor antagonist losartan. A final set of experiments suggests activation of PVN neurons during hyperinsulinemia increases glutamatergic drive to the rostral ventrolateral medulla. Collectively, these findings indicate that insulin activates a melanocortin-dependent pathway to the PVN that increases glutamatergic drive to the rostral ventrolateral medulla and alters cardiovascular function.

Median preoptic neurones projecting to the hypothalamic paraventricular nucleus respond to osmotic, circulating Ang II and baroreceptor input in the rat

The present study sought to determine whether individual neurones of the median preoptic nucleus (MnPO) with axonal projections to the hypothalamic paraventricular nucleus (MnPO‐PVN) respond to osmotic, circulating angiotensin II (Ang II), and baroreceptor stimulation. Hypertonic NaCl (0.75 or 1.5 osmol l−1) or Ang II (150 ng) was injected into the internal carotid artery (ICA). Baroreceptor stimulation was performed by i.v. injection of phenylephrine or sodium nitroprusside to increase or decrease arterial blood pressure, respectively. Of 65 MnPO neurones, 50 units were antidromically activated from the PVN with an average onset latency of 11.3 ± 0.7 ms. Only 9.5% of MnPO‐PVN neurones were antidromically activated from the PVN bilaterally. Type I MnPO‐PVN neurones (n= 14) responded to osmotic but not Ang II stimulation. In 79% (11/14) of these type I neurones, the response was an increase in cell discharge. Type II MnPO‐PVN neurones (n= 7) displayed a significant increase in cell discharge in response to ICA injection of Ang II but not hypertonic NaCl. Type III MnPO‐PVN neurones (n= 16) responded to both ICA injection of hypertonic NaCl and Ang II. In 88% (14/16) of type III neurones, osmotic and Ang II stimulation each increased cell discharge. Type IV MnPO‐PVN neurones (n= 13) displayed no change in cell discharge in response to ICA injection of hypertonic NaCl or Ang II. Baroreceptor stimulation altered the discharge in subpopulations of type I, II and III MnPO‐PVN neurones (43–63% depending on neuronal type). Only one MnPO‐PVN neurone responded solely to baroreceptor stimulation (type IV). In addition, a subset of type I, II and III neurones displayed a significant correlation with sympathetic nerve activity and/or the cardiac cycle. These findings suggest that a significant population of MnPO‐PVN neurones respond to osmotic and circulating Ang II stimulation and thereby represents a neural substrate through which neurohumoral inputs are integrated within the forebrain lamina terminalis.

Hypothalamic paraventricular nucleus differentially supports lumbar and renal sympathetic outflow in water‐deprived rats

The present study sought to determine whether the hypothalamic paraventricular nucleus (PVN) contributes in a time‐dependent manner to the differential patterning of lumbar and renal sympathetic nerve activity (SNA) in water‐deprived rats. Mean arterial blood pressure (MAP) and both lumbar SNA (LSNA) and renal SNA (RSNA) were recorded simultaneously in control, 24 and 48 h water‐deprived rats, and the PVN was inhibited bilaterally with microinjection of the GABAA agonist muscimol (100 pmol in 100 nl per side). Inhibition of the PVN significantly decreased RSNA in 48 h water‐deprived rats but not in 24 h water‐deprived or control rats (48 h, −17 ± 4%; 24 h, −2 ± 5%; control, 4 ± 6%; P < 0.05). In addition, injection of muscimol significantly decreased LSNA in 48 and 24 h water‐deprived rats but not in control rats (48 h, −41 ± 4%; 24 h, −14 ± 6%; control, −3 ± 2%; P < 0.05). Interestingly, the decrease in LSNA was significantly greater than the decrease in RSNA of 24 and 48 h water‐deprived rats (P < 0.05). Inhibition of the PVN also significantly decreased MAP to a greater extent in 48 and 24 h water‐deprived rats compared to control rats (48 h, −34 ± 5 mmHg; 24 h, −26 ± 4 mmHg; control, −15 ± 3 mmHg; P < 0.05). When 48 h water‐deprived rats were acutely rehydrated by giving access to tap water 2 h before experiments, inhibition of the PVN with muscimol did not alter LSNA (−12 ± 8%) or RSNA (7 ± 4%) but did produce a small decrease in MAP (−15 ± 4 mmHg) that was not different from control rats. In a parallel set of experiments, acute rehydration of 48 h water‐deprived rats significantly attenuated the increased Fos immunoreactivity in PVN neurones that project to the spinal cord or rostral ventrolateral medulla. Collectively, the present findings suggest that PVN autonomic neurones are synaptically influenced during water deprivation, and that these neurones differentially contribute to LSNA and RSNA in water‐deprived rats.

Hyperosmotic activation of CNS sympathetic drive: implications for cardiovascular disease

Evidence now indicates that exaggerated sympathetic nerve activity (SNA) significantly contributes to salt‐sensitive cardiovascular diseases. Although CNS mechanisms that support the elevation of SNA in various cardiovascular disease models have been intensively studied, many mechanistic details remain unknown. In recent years, studies have shown that SNA can rise as a result of both acute and chronic increases of body fluid osmolality. These findings have raised the possibility that salt‐sensitive cardiovascular diseases could result, at least in part, from direct osmosensory activation of CNS sympathetic drive. In this brief review we emphasize recent findings from several laboratories, including our own, which demonstrate that neurons of the forebrain organum vasculosum laminae terminalis (OVLT) play a pivotal role in triggering hyperosmotic activation of SNA by recruiting neurons in specific regions of the hypothalamus, brainstem and spinal cord. Although OVLT neurons are intrinsically osmosensitive and shrink when exposed to extracellular hypertonicity, it is not yet clear if these processes are functionally linked. Whereas acute hypertonic activation of OVLT neurons critically depends on TRPV1 channels, studies in TRPV1−/− mice suggest that acute and long‐term osmoregulatory responses remain largely intact. Therefore, acute and chronic osmosensory transduction by OVLT neurons may be mediated by distinct mechanisms. We speculate that organic osmolytes such as taurine and possibly novel processes such as extracellular acidification could contribute to long‐term osmosensory transduction by OVLT neurons and might therefore participate in the elevation of SNA in salt‐sensitive cardiovascular diseases.

Neurons of the Rostral Ventrolateral Medulla Contribute to Obesity-Induced Hypertension in Rats

Activation of the sympathetic nervous system contributes to the pathogenesis of obesity-induced hypertension. The present study sought to determine whether sympathetic regulatory neurons of the rostral ventrolateral medulla contribute to the elevated blood pressure in obese rats. Male Sprague–Dawley rats (350 to 425 g) were placed on a moderately high-fat diet (32% kcal as fat) or a low-fat (LF) diet (10.6% kcal as fat). After 13 weeks, rats fed the moderately high-fat diet segregated into obesity-prone (OP) and obesity-resistant (OR) groups based on their body weight (OP: 839±22 g; OR: 668±15 g; LF: 680±18 g; n=15 for all groups; P<0.01). Under isoflurane anesthesia, baseline mean arterial blood pressure was significantly elevated in the OP rats versus the OR and LF rats (OP: 108±2 mm Hg; OR: 100±2 mm Hg; LF: 97±3 mm Hg; n=7; P<0.05). Inhibition of the rostral ventrolateral medulla with bilateral microinjection of the GABAA receptor agonist muscimol (200 pmol/100 nL) decreased mean arterial blood pressure to similar levels across the groups (OP: 49±1 mm Hg; OR: 50±2 mm Hg; LF: 49±1 mm Hg), but the magnitude of this decrease was significantly greater in the OP versus the OR and LF rats (OP: −58±2 mm Hg; OR: −49±1 mm Hg; LF: −48±3 mm Hg; P<0.01). These differences in mean arterial blood pressure cannot be explained by changes in vascular reactivity as the ED50 in response to phenylephrine and norepinephrine was similar across the groups. The present findings suggest that the elevated sympathetic nerve activity and arterial blood pressure in obese rats depends on the tonic activity of rostral ventrolateral medulla sympathetic neurons.

Transneuronal tracing of neural pathways controlling activity of diaphragm motoneurons in the ferret.

Previous studies have shown that neurons in addition to those in the medullary respiratory groups are involved in activating phrenic motoneurons during a number of behaviors, including vomiting and reaction to vestibular stimulation. However, the location of premotor inspiratory neurons outside of the main medullary respiratory groups is largely unknown, particularly in emetic species. In the present study, the transneuronal tracer pseudorabies virus was injected into the diaphragm of the ferret, and the locations of retrogradely-labeled motoneurons and transneuronally-labeled pre-motoneurons in the brainstem and cervical and thoracic spinal cord were mapped. Injections of a monosynaptic tracer, cholera toxin, were also made in order to verify the location of motoneurons innervating the diaphragm. Phrenic motoneurons identified with pseudorabies virus and cholera toxin were confined largely to the C5-C7 levels of spinal cord, and often gave rise to prominent polarized dendritic arbors that extended across the midline. At post-inoculation survival times > or = three days, transneuronally-labeled interneurons were located in the cervical and thoracic spinal cord and portions of the brainstem, including the midline pontomedullary reticular formation and the lateral medullary reticular formation. Double-labeling studies revealed that although the infected midline neurons were located in the proximity of serotonergic neurons, only a small number of the virus-containing cells were positive for serotonin. These findings suggest that neurons in the midline of the medulla and pons influence the activity of phrenic motoneurons, perhaps during inspiratory behaviors unique to emetic animals (such as vomiting).

Increased Dietary Salt Enhances Sympathoexcitatory and Sympathoinhibitory Responses From the Rostral Ventrolateral Medulla

Increased dietary salt exaggerates arterial blood pressure (ABP) responses evoked from the rostral ventrolateral medulla (RVLM). The present study determined whether these enhanced pressor responses were directly attributable to a greater increase in sympathetic nerve activity (SNA) and whether these enhanced responses were balanced by a greater responsiveness of RVLM neurons to inhibitory input. Male Sprague-Dawley rats were fed normal chow and given access to either water or a 1% NaCl solution for 14 days. Injection of l-glutamate (0.03, 0.1, 1.0, and 3.0 nmol) into the RVLM produced a significantly greater increase in renal SNA, splanchnic SNA, and ABP in rats drinking 1% NaCl versus water. Conversely, injection of the inhibitory amino acid &ggr;-aminobutyric acid (0.1, 1.0, and 10 nmol) into the RVLM produced significantly greater decreases in renal SNA, splanchnic SNA, and ABP of rats drinking 1% NaCl versus water. These enhanced SNA and ABP responses to l-glutamate and &ggr;-aminobutyric acid were not observed in rats drinking 1% NaCl for 1 or 7 days but were present in rats drinking 1% NaCl for 21 days. Moreover, the dietary salt-induced enhancement of both sympathoexcitatory and sympathoinhibitory responses from the RVLM persisted after the 1% NaCl solution was replaced with water for 1, but not 7, days. These findings indicate that the potentiated ABP responses observed previously are mediated by parallel changes in SNA, and these responses depend on a slowly developing and reversible form of neuronal plasticity.

Glutamatergic Receptor Activation in the Rostral Ventrolateral Medulla Mediates the Sympathoexcitatory Response to Hyperinsulinemia

Hyperinsulinemia increases sympathetic nerve activity (SNA) and has been linked to cardiovascular morbidity in obesity. The rostral ventrolateral medulla (RVLM) plays a key role in the regulation of SNA and arterial blood pressure (ABP). Many sympathoexcitatory responses are mediated by glutamatergic receptor activation within the RVLM, and both the central renin–angiotensin and melanocortin systems are implicated in the sympathoexcitatory response to hyperinsulinemia. Therefore, we hypothesized that one or more of these neurotransmitters in the RVLM mediate the sympathoexcitatory response to insulin. Hyperinsulinemic–euglycemic clamps were performed in &agr;-chloralose anesthetized, male Sprague–Dawley rats by infusion of insulin (3.75 mU/kg per minute, IV) and 50% dextrose solution for 120 minutes. Physiological increases in plasma insulin elevated lumbar SNA, with no change in renal SNA, ABP, or blood glucose. Microinjection of the ionotropic glutamate receptor antagonist kynurenic acid into the RVLM significantly reduced lumbar SNA and ABP. Selective blockade of NMDA but not non-NMDA glutamate receptors resulted in similar reductions of lumbar SNA. In marked contrast, microinjection of the angiotensin II type 1 receptor antagonist losartan or the melanocortin 3/4 antagonist SHU9119 had no effect on lumbar SNA or ABP. Western blot analysis showed that insulin receptor expression is significantly lower in the RVLM than the hypothalamus, and direct microinjection of insulin into the RVLM did not significantly increase lumbar SNA. These findings suggest that hyperinsulinemia increases lumbar SNA by activation of a glutamatergic NMDA-dependent projection to the RVLM.

Mice lacking the transient receptor vanilloid potential 1 channel display normal thirst responses and central Fos activation to hypernatremia.

Neurons of the organum vasculosum of the lamina terminalis (OVLT) are necessary for thirst and vasopressin secretion during hypersmolality in rodents. Recent evidence suggests the osmosensitivity of these neurons is mediated by a gene product encoding the transient receptor potential vanilloid-1 (TRPV1) channel. The purpose of the present study was to determine whether mice lacking the TRPV1 channel had blunted thirst responses and central Fos activation to acute and chronic hyperosmotic stimuli. Surprisingly, TRPV1-/- vs. wild-type mice ingested similar amounts of water after injection (0.5 ml sc) of 0.5 M NaCl and 1.0 M NaCl. Chronic increases in plasma osmolality produced by overnight water deprivation or sole access to a 2% NaCl solution for 48 h produced similar increases in water intake between wild-type and TRPV1-/- mice. There were no differences in cumulative water intakes in response to hypovolemia or isoproterenol. In addition, the number of Fos-positive cells along the lamina terminalis, including the OVLT, as well as the supraoptic nucleus and hypothalamic paraventricular nucleus, was similar between wild-type and TRPV1-/- mice after both acute and chronic osmotic stimulation. These findings indicate that TRPV1 channels are not necessary for osmotically driven thirst or central Fos activation, and thereby suggest that TRPV1 channels are not the primary ion channels that permit the brain to detect changes in plasma sodium concentration or osmolality.